Due to recent advances in DNA sequencing techniques, the sequencing of viral DNAs is limited only by the availability of sufficiently closely spaced specific priming sites for DNA polymerase. We will therefore perfect and apply a new very rapid method for the mapping of DNA restriction fragments. Such mapping restriction fragments provide ideal primers for the new sequence methods. Our mapping method, based on a two-dimensional nucleic acid hybridization procedure, will be used to map bacteriophage phi X174 DNA using a number of newly discovered restriction enzymes. We will also attempt to use the method to map phi X174 mRNAs with respect to the restriction map. As another approach to the sequencing problem, we will explore the use of short synthetic oligonuleotides as specific primers for sequence determination. We will attempt to develop general and practical methods for the construction of mutants containing specific predetermined alterations and within known nucleotide sequences. This will be done by: 1) introducing mutagenic nucleotide at specific points in the sequence using enzymatic techniques and 2) the incorporation of synthetic oligonucleotide, containing a mutant sequence into the phi X174 genome.